wilson method for the bacteriological examination of water

Vol. 30

Wilson Method for the BacteriologicalExamination of Water

RAUL FERRAMOLALaboratories of Obras Sanitarias de la Naci6n, Buenos Aires, Argentine

AMONG the numerous methods pro-posed for determining the coli-

form count of water the two mostwidely used are those of the AmericanPublic Health Association and theBritish Ministry of Health.

In the American Standard Methodthe water is inoculated into lactosebroth; tubes showing gas are plated onEndo or eosine-methylene-blue agar;and one or more typical colonies arepicked off into lactose and studiedmicroscopically to see whether the con-tained organisms have the morphologi-cal and staining properties of coliformbacilli. By this means it is possibleto determine the number of coliformbacilli present in the water, but not toform any idea as to whether these or-ganisms belong to the fecal coli, theintermediate-aerogenes-cloacae, or ir-regular type.*

In the British Ministry of Health(1934) method, lactose broth is re-placed by MacConkey's broth. Since

*EDITOR'S NOTE: The classification of coliformorganisms employed by the author is that of theBritish Ministry of Health. The group designatedas " Pecal coli, or Bact. col I " comprises thosecoliform organisms that give positive results with themethyl red, Voges-Proskauer, indol tests and gas inMacConkey broth at 440 C., but negative results withthe citrate and the gelatin liquefaction 7 day tests.The group " Bact. coli 1 " comprises those coliformsthat give a positive methyl red test, but negativeresults with all the other tests; their probablehabitat is indicated as " doubtful, probably notprimarily intestinal." The other designations arerather similar to those used in America, but com-plete details may be found in author's Reference No;4, or J. Am. Water Works Assn., 31, 7:1233 (July),1939.

this medium has a high degree ofselectivity on account of the incorpora-tion of 0.5 per cent bile salt, it is usualto accept the presumptive coliformcount obtained in this way as the realcoliform count. Differentiation, how-ever, of the coliform bacilli is advisedin certain circumstances which arecarefully defined. This is carried outby plating one or more of the positivetubes on MacConkey agar, picking offthree colonies from each plate, and sub-mitting them to the methyl red (M.R.),Voges-Proskauer (V.P.), indol, andcitrate tests.

In practice the American. methodsuffers from the disadvantage that thepresumptive coliform count in lactosebroth is accompanied by a large errordue to false positive reactions, and nodifferentiation is made between the,types of coliform bacilli present. Th-Ministry of Health method, though freefrom these particular- disadvantages,neverth-eless uses a method of differen-.tiation which is itself open to objection..

In the first place, if the various types.of coliform bacilli differ greatly in num-ber, and plates are made only from thetubes inoculated with the smallestquantities of water, the probability isthat only one type will be revealed.

In the second place, even if the vari-.ous types are more or less equal innumber, it is very common for Bact.^coli to outgrow Bact. aerogenes and,other types in the MacConkey broth'

[10831

AMERICAN JOURNAL OF PUBLIC HEALTH

tubes. Consequently plates made fromthe fermented tubes often reveal a pure

culture of Bact. coli, even when othercoliform types were present originally.The colonies on the plates correspond,in fact, not to the types present in theoriginal water, but to types present inthe fermented MacConkey broth tubes.To overcome the first disadvantage,

it would be necessary to plate out every

positive tube on MacConkey agar or

other suitable medium, and to submitthree or more colonies from each plateto the M.R., V.P., indol, and citratetests. Though possible with some

waters, this would often involve more

labor and cost than could well beafforded in practice.To overcome the second disadvan-

tage, nothing but the use of highlyselective media which would allow thedevelopment of different types of or-

ganisms, even if present in minimalnumbers, would be of any value.An attempt to overcome these two

difficulties was made by Wilson and hiscolleagues (1935) in London, whenengaged in a study of the bacteriologicalgrading of milk. The method they de-vised, which is referred to as MethodIV, consists essentially in the use oftwo selective media, one for thedemonstration of Bact. coli I (seeTable III), the other for the demonstra-tion of the intermediate, Bact. aero-

genes, and Bact. cloacae types (I.A.C.).In practice the water is inoculated

into MacConkey broth, as in the firststep in the Ministry of Health method,so as to obtain the presumptive coliformcount. All tubes showing acid and gaswithin 48 hours are inoculated into (1)tubes of MacConkey broth, and (2)tubes of Koser's citrate medium. TheMacConkey broth tubes are incubatedat 440 C. in a thermostatically con-trolled water bath; under these con-

ditions with few expections, only strainsof fecal coli give rise to gas formation.The citrate tubes are incubated at

370 C.; in this medium strains ofI.A.C. type, but not usually Bact. coliI, develop. The 440 C. MacConkeytubes showing gas formation afford anindex of the probable number of fecalcoli, and the citrate tubes of theprobable number of I.A.C. types presentin the water.

This method is. very simple andrapid. It enables not only a presump-tive coliform count, but a differentialcount, to be completed within 3 or 4days. It has been tried out on waterwith very favorable results by Bardsley(1938) at Manchester and, togetherwith suggested modifications, is de-scribed in the latest edition of theMinistry of Health memorandum(1939) on the bacteriological examina-tion of water supplies. Referenceshould be made to this publication fora full description of the method and itsinterpretation.

Since learning of this method inEngland, we have examined about10,000 samples of water in the Argen-tine with highly satisfactory results.In addition, we have carried out cer-tain controls of the main steps of theWilson method. These we propose todescribe in the following pages.

CONTROL OF THE PRESUMPTIVE COLI-FORM COUNT IN WATER USINGMAC CONKEY BROTH AT 370 C.

Tubes of MacConkey broth showingacid and gas formation at 370 C.,which had been seeded with the smallestquantity of water, were plated onMacConkey or eosine-methylene-blueagar, and incubated for 24 hours.Coliform-like colonies were picked off,and studied for their morphological andstaining properties and for their abilityto form gas in standard lactose broth.They were also examined by the 44° C.MacConkey and the citrate tests. Inthis way 430 fermented MacConkeybroth tubes were checked. The resultsare given in Table 1.

Sept., 19401084

EXAMINATION OF WATER

TABLE 1

The Accuracy of the Presumptive Coliform Count in MacConkey Broth at 370 C.

Fermented MacConkey Broths

24 Hoursr

PercentageControlled Confirmed Confirmed

77

19

96

48 Hours

PercentageControlled Confirmed Confirmed

77 100 231 205

16

93

84

97

103

334

81

286

89

79

86Total controlled (24 and 48 hours) ...... 430

Total confirmed (24 and 48 hours) ...... 379

Percentage confirnmed (24 and 48 hours).. 88

It will be noticed that the percentageof cultures confirmed was (1) muchhigher with tubes showing fermentationwithin 24 hours than in those taking48 hours, and (2) much higher withsurface than with underground waters.

In considering this second observa-tion it may be pointed out that themajority of the surface waters camefrom Buenos Aires, and were examinedwithin a few hours, whereas all theunderground waters came from variousparts of the Argentine outside BuenosAires, and were not examined until, insome instances, 2 days later. This cir-cumstance may also be partly respon-sible for the fact that 40 out of the 51false positive tubes contained coliform-like bacilli which failed to producelactose-fermenting colonies on plateswithin 24 hours. It seems not im-probable that many of these organismswere in reality coliform bacilli whosefermenting properties had become modi-fied by the unfavorable conditions oftransport. In support of this explana-tion is the fact that most of these or-ganisms were derived from pollutedwaters which had been chlorinated.Some authors would ascribe the samesanitary importance to these organismsas to those of the coli-aerogenes group.Of the remaining false positive tubes,

5 consisted of coliform bacilli whichwere typical except in their failure toferment lactose at 370 C.; all of theseproduced gas in MacConkey broth at440 C. In 6 false positive tubes nocoliform bacilli could be demonstratedby plating, though Gram-positive rodswere sometimes present.Thus it appears that all but 6 of the

false positive tubes contained bacilliwhich differed from coliform bacilli onlyin their failure to ferment lactosewithin 24 hours at 370 C., either onplates or in broth. False positives dueto other types of organism were veryfew, and accounted for only 1.4 percent of the whole series.The high proportion of correct results

obtained with MacConkey's medium isin agreement with the findings of manyother workers who have had extensiveexperience of this medium, such asBardsley (1938) in England, andRaghavachari and Iyer (1936) inIndia. It may be noted that from 550waters producing acid and gas inMacConkey broth at 370 C. Bardsleywas able to isolate coliform bacilli inevery instance. Not a single falsepositive due to non-coliform bacilliwas encountered.*

* EDITOR'S NOTE: See also this Journal, 29, 11,

1250 (Nov.), 1939.

Origin ofWater SamplesSurface

(rivers)Underground

(wells)

Total

Vol. 30 108Si


CONTROL OF THE FECAL COLI COUNT INWATER USING MAC CONKEY

BROTH AT 440 C.As in the previous experiments we

examined tubes of MacConkey brothshowing acid and gas at 440 C. byplating on MacConkey or eosine-methylene-blue agar, picking 3 colonies,and testing them by the M.R., V.P.,citrate, and indol tests. The 440 C.MacConkey broths had been inoculateddirectly from fermented 370 C. Mac-Conkey broths which had been seededwith the smallest quantity of water.In this way we checked 309 positive440 C. tubes with the results shown inTable 2.

ing 6 no growth was obtained on solidmedia. In other words, the 440 C.MacConkey test yielded correct resultsin at least 95 per cent of tubes.Of practical interest is the fact that

95 per cent of the tubes fermentingMacConkey broth at 440 C. did sowithin 24 hours, thus enabling differen-tiation to be made in 2 to 3 days fromthe primary inoculation of the water.

CONTROL OF THE SPECIFICITY OF THE440 C. MAC CONKEY TEST FOR FECAL

COLI IN SEWAGE AND FECESSewage from Buenos Aires and

animal feces from various sources wereplated on eosine-methylene-blue agar

TABLE 2The Accuiracy of the Fecal Coli Count in MacConkey Broth at 440 C.-Water

440 C. MacConkey Broths Inoculated from MacConkeyBroths That Had Fermented at 370 C. in

24 Hours 48 Hours

PercentageControlled Confirmed Confirmed Controlled Confirn

205 199 97 49 47

42

247

40

239

95

97

13

62

11

58

Percentagemed Confirmed

96

85

94

Of the 309 tubes fermenting Mac-Conkey broth at 440 C., 294 yieldedBact. coli I (fecal), 3 Bact. coli II, and6 intermediate, Bact. aerogenes, or ir-regular strains, while from the remain-

and incubated for 24 hours at 370 C.Typical colonies were picked off andtested by the M.R., V.P., citrate, indol,and 440 C. MacConkey broth tests.The organisms were grouped according

TABLE 3The Specificity of the 440 C. MacConkey Broth Test for Fecal Coli: Sewage and Feces

Feces from

Sewage Sparrow Pigeon Vigud Cattle

No. of Positive No. of Positive No. of Positive No. of Positive No. of PositiveType of Organism Strains at 440 C. Strains at 440 C. Strains at 440 C. Strains at 440 C. Strains at 440 C.Bact. coli type I 116 115 85 85 198 198 60 60 504 499Bact. coli type II 3 1 0 0 1 1 0 0 0 0Intermediate type I 34 0 0 0 0 0 0 0 0 0Intermediate type II 26 0 0 0 0 0 0 0 0 0Bact. aerogenes type I 40 0 0 0 1 0 0 0 1 0Bact. aerogenes type II 3 0 0 0 0 0 0 0 0 0Bact. cloacae 0 0 0 0 0 0 0 0 0 0

1 The Vigua (Phalacrocorax olivaceus, Humboldt) is a bird, similar to the Brazilian Cormiiorant, that iscommon over the settling basins of water purifying plants.


(rivers)Underground

(wells)

Total

1086 Sept., 1940


to the classification given by theMinistry of Health (1939). Alto-gether 222 colonies from sewage and850 colonies from feces were examined.The results are recorded in Table 3.Of 963 strains classified as Bact.

coli I, all but 6 produced gas inMacConkey broth at 440 C., while of105 other coliform strains, excluding 4strains of Bact. coli II the fecal originof which is in some doubt, not a singleone did so. It would be unreasonableto demand a greater degree of specificityfor any test.The 440 C. MacConkey broth test

thus affords a rapid and accuratemethod for differentiating Bact. coli I,the typical inhabitant of the intestine,from other coliform organisms whoseprimary habitat appears to be most fre-quently outside the animal body.

CONTROL OF THE INTERMEDIATE-AEROGENES-CLOACAE COUNT IN WATER

USING KOSER'S CITRATE AT 370 C.Positive cultures in Koser's citrate

medium, which had been seeded fromfermenited MacConkey broth tubesinoculated with the smallest quantityof water, were plated on eosine-methylene-blue agar. Three colonieswere picked off 24 hours later andstudied in the usual way. The resultsare recorded in Table 4.A considerable difference was no-

ticeable between citrate-positive tubes

The Specificity


(rivers)Underground

(wells)

Total

64

36

100

33

92

inoculated from 370 C. MacConkeybroths that had fermented within 24hours and those that had taken 48hours. In 92 per cent of the formercoliform bacilli were found, but in only61 per cent of the latter. The reasonfor this difference is not clear.

Examination of the colonies that de-veloped on plates streaked from thefalse positive citrate tubes showed themto consist of non-lactose-fermentingGram-negative coliform-like bacilli.What were these organisms? Werethey non-lactose or late lactose-fermenting bacilli which were presentin the original water, and which de-veloped particularly during the secondday of incubation of the 370 C.MacConkey broths, or were they or-ganisms of the I.A.C. group that hadtemporarily lost their power of fer-menting lactose within 24 hours?Our observations were not suffi-

ciently extensive to answer these ques-tions, but it seems probable that manyof the organisms belonged to the secondclass. For example, analysis of 142false citrate positives showed that 108had been inoculated from 370 C. Mac-Conkey broths which had failed onsubculture to produce gas in Mac-Conkey broth at 440 C. In otherwords, the 108 370 C. MacConkeybroths contained no fecal coliformorganisms, and the fermentation of lac-tose in these tubes must have been due

TABLE 4

of Koser's Citrate Medium for Organisms of the I.A.C. Group-WaterPositive Citrate Tubes Inoculated from 370 C. MacConkey Broths

Showing Fermentation at

48 Hours

PercentageConfirmed Confirmed

86 57

191

342

122

208

64

61

92

92

24 Hours

PercentageControlled Confirmed Confirmed Controlled

59 92 151

Vol. 30 1087


to organisms of the I.A.C. group, or tosome non-coliform type of organism.Since examination of Table 1 showsthat the total proportion of false posi-tives in 370 C. MacConkey broth wasonly 12 per cent, it is difficult to avoidthe conclusion that the great majorityof the positive citrate tubes must havecontained coliform bacilli, even thoughtheir presence could not be demon-strated by direct plating.

It would therefore appear that therelatively high proportion of false posi-tives in Koser's citrate medium seen inTable 4 is more apparent than real, andthat the specificity of the citrate testfor organisms of the I.A.C. group,while not so high as that of the 44° C.MacConkey test for fecal coli, is never-theless of a reasonably high order.

It may be noted that, though fecalcoli was found on some plates madefrom positive citrate tubes, it was in-variably accompanied by members ofthe I.A.C. group. That is to say, nofalse positives in citrate were due tofecal coli.


unchlorinatedUnderground

unchlorinated

Total

Surfacechlorinated

Undergroundchlorinated

Total

Grand Total

COMPARATIVE STUDY OF THE AMERICANSTANDARD AND THE WILSON METHODSFinally we should like to refer to a

comparative examination made on 499waters by the American Standard andthe Wilson methods. The samples werecollected in Buenos Aires and in otherparts of the Argentine. About 70 percent were from chlorinated supplies.

In recording the results of the Wilsonmethod, we have confined ourselves tothe coliform count, since it is this alonewhich is common to both methods. Thedifferential counts of fecal coli andI.A.C. organisms, which were obtainedby the Wilson method, are omitted fromTable 5, though the number of tubesgiving a positive 440 C. MacConkeyand a positive citrate test are includedfor the sake of interest.On the whole, the number of samples

in which the presence of coliformbacilli was confirmed was remarkablysimilar in the two methods. The num-ber of discrepant results was 39 in atotal of 3,245 tubes inoculated, or justover 1 per cent.

TABLE 5

Comparison of the American Standard and the Wilson Methods

American Standard ColiformMethod Wilson Method Organisms

>: + NA t9 0 +;g @) l0 '0It

111 569 292 198 (68) 228 169 187

45 315

156 884

294 1,998

49 363

343 2,361

499 3,245

43

335

31 (72)

229 (68)

44 18 31

272 187 218

864 119 (14) 140 95 74

39

903

1,238

8 (20)

127 (14)

356 (29)

11 2

151 97

423 284

10

84

302

200 (88)

36 (82)

236 (87)

4 2

6 1

10 3

128 (91) 15

9 (82)

137 (91)

373 (88)

6

2

8

11

3

18

28

-1088 Sept., 1940


The number of positive tubes was alittle higher by the Wilson method, sug-gesting a slightly greater degree ofsensitivity. The reason for this ap-peared to be due mainly to the failure,in the American Standard method, oftypical lactose-fermenting colonies todevelop on eosine-methylene-blue agarplates within 24 hours. Two causesseemed to be responsible for this: (1)In some waters containing slow lactose-fermenting organisms, such as in certainwell waters, the colonies on plates ex-amined after 24 hours were still color-less, though when incubated for afurther 1 to 2 days they developed intotypical lactose fermenters. (2) In somewaters organisms of the Pseudomonasgroup were common, and these over-grew the coliform organisms on platesand prevented their developing intotypical lactose-fermenting colonies. Someof the positive results given by theWilson method may, of course, havebeen caused by the growth of non-coliform organisms in citrate, thoughthese do not appear to have beennumerous.

Analysis showed that in all but oneof the 39 discrepant results, theaverage number of coliform organismswas only 2.2 per 100 ml. In rela-tively pure waters of this type thesampling error alone may well havebeen sufficient to account for many ofthe discrepancies observed.

It is interesting to note that a verysmall proportion of fermented lactosebroth tubes in the American Standardmethod were confirmed on plating.With chlorinated waters, for example,this proportion was only 14 per cent.Analysis of the figures obtained withthe Buenos Aires waters, which cannotbe given in detail here, showed thatthe proportion was lowest of all withfreshly chlorinated waters. In oneseries of 77 fermented lactose brothsinoculated from such waters only onetube was confirmed.

DISCUSSIONFrom the results given in this paper

it will be realized that the Wilsonmethod of examining waters is veryrapid and affords an assessment notonly of the coliform bacilli present, butalso of the proportions of Bact. coli onthe one hand, and of the intermediate,Bact. aerogenes, and Bact. cloacaetypes on the other.The 440 C. MacConkey broth test,

provided it is carried out with the pre-cautions laid down in the Ministry ofHealth Report (1939), serves as a mostreliable, delicate, and rapid index ofthe presence of fecal coli.The use of the citrate test for de-

tecting organisms of the I.A.C. group isless specific, but since the occurrenceof these organisms in the concurrentpresence of fecal coli is of little help ininterpreting the results of the water ex-amination, and since their occurrence inthe absence of fecal coli may be re-garded as indicative of no immediatedanger, and a confirmatory plating testmay be made if necessary, this draw-back is of less importance than mightappear at first sight.The fact that the Wilson methbd

enables a confirmed and a differentialcoliform count to be completed in thesame time as that required by theAmerican Standard method for thecoliform count alone, and the fact thatit dispenses with plating and withmicroscopical examination, are two ad-vantages which justify its trial on amore extensive scale.

SUMMARY AND CONCLUSIONS1. An attempt has been made to confirm

the accuracy of the various steps used in theWilson method of performing the coliformand differential coliform counts on water.This method, which is simple and rapid, con-sists in the use of MacConkey broth at 370 C.for the presumptive coliform count, followedby subculture of all fermented tubes intoMacConkey broth at 440 C. for the fecal colicount and into Koser's citrate at 370 C. forthe intermediate-aerogenes-cloacae count.

Vol. 30 1089

1090 AMERICAN JOURNAL OF PUBLIC HEALTH Sept., 1940

2. Control of the presumptive coliformcount in MacConkey broth at 370 C. byplating and examination of the resultantcolonies was carried out on 430 fermentedtubes inoculated from surface and under-ground waters. Of tubes showing fermenta-tion within 24 hours, 97 per cent were con-firmed, while of those not showing fermenta-tion until the second day, 86 per cent wereconfirmed. The fact that the proportionswere higher in samples of Buenos Aireswaters, which were examined on the sameday, than in waters from other parts of theArgentine, which sometimes required 1 or 2days to reach the laboratory, suggests thatthe ability of contained organisms to formlactose-fermenting colonies on plates in 24hours may have been affected by the unfavor-able conditions of transport.

3. Control of the fecal coli count inMacConkey broth at 440 C. in water wasmade by plating out 309 fermented tubes,picking off colonies, and ascertaining to whichtype they belonged by the usual differentialtests. It was found that 294 containedtypical Bact. coli I, while 3 contained Bact.coli II. At least 95 per cent, therefore, ofthe results were confirmed.

4. Further control of the 440 C. MacConkeytest was made by plating out sewage, animalfeces, and bird feces, picking off lactose-fermenting colonies, ascertaining their type bythe usuaI differential tests, and inoculatingthem into MacConkey broth at 440 C. Of atotal of 963 colonies classified as Bact. coli I,all but 6 produced gas in this medium, whileof 105 other coliform strains, excluding 4strains of Bact. coli II, the fecal origin ofwh6ch is in some doubt, not a single one didso. The error of the 440 C. MacConkey brothtest as an index of fecal coli was thereforeless .than 1 per cent.

5. The Koser citrate test at 370 C. for de-tecting the presence of coliform organisms ofthe I.A.C. group in water was controlled byinoculating 442 fermented 370 C. MacConkeybroth tubes into citrate, plating out all tubesshowing growth, and studying the colonies inthe usual way. Of positive citrate tubes thathad been inoculated from 370 C. MacConkeybroths fermenting in 24 hours, 92 per centwere shown to contain coliform bacilli,whereas of those inoculated from MacConkey

broths not fermenting until the second day,only 61 per cent were confirmed. A largeproportion of the false positives in citratemedium were due apparently to the failureof coliform bacilli to produce typical lactose-fermenting colonies or to ferment lactose brothwithin 24 hours. In other words, the rela-tively high proportion of false positives wasprobably more apparent than real.

6. A comparison was made between theAmerican Standard and the Wilson methodson 499 waters, paying attention merely to theconfirned coliform count. The results, on thewhole, were in close agreement. In a seriesof 3,245 tubes inoculated with water, the finaldiscrepancies were only 39 in number, or alittle over 1 per cent. The Wilson methodwas positive 28 times when the AmericanStandard method was negative, and the latterwas positive 11 times when the Wilsonmethod was negative. In all but one of the39 discrepant results, the waters were of arelatively high degree of purity, containing anaverage of only 2.2 coliform bacilli per 100 ml.

7. In view of the simplicity and accuracyof the Wilson method, and the fact that itgives a differential as well as a confirmed coli-form count in the same time as that requiredby the American Standard method to give acoliform count alone, the method seemsworthy of trial on a more extensive scale.During the year 1938 we examined about10,000 samples of water by this method withhighly satisfactory results.

ACKNOWLEDGMENTS-We wish to thankProfessor G. S. Wilson for help and adviceduring the time we spent at the LondonSchool of Hygiene and Tropical Medicine, andMiss D. Bardsley, with whom we practisedthe new method in the Department of Bac-teriology and Public Health at ManchesterUniversity.

REFERENCES1. Bardsley, Doris A. J. Hyg., 38, 309-324, 1938.2. Raghavachari, T. N. S., and Iyer, P. V. S.

Indian J. M. Research, 23, 619-666, 1936.3. Report. Rep. Publ. Hlth. and Med. Subj., MiX.

Hlth., London, No. 71, 1934.4. Report. Ibid., 1939.S. Wilson, G. S., Twigg, R. S., Wright, R. C.,

Hendry, C. B., Cowell, M. P., and Maier, I. Spec.Rep. Ser., Med. Res. Council, London, No. 206,1935.

wilson method for the bacteriological examination of water

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